Abstract This proposal concerns the molecular and cellular mechanisms that determine synaptic connectivity in developing nervous systems. It focuses on a set of Drosophila neuronal cell surface/signal transduction proteins that we have been studying for many years: the transmembrane receptor tyrosine phosphatases (RPTPs). The six Drosophila RPTPs are central regulators of axon guidance and synaptogenesis. They have been extensively investigated using genetics, but we still known relatively little about the signaling pathways in which they function. This proposal is directed toward identification and characterization of components of these pathways. Specific aims 1 and 2 concern new methods for identification of RPTP ligands and coreceptors. We have already defined a Ptp10D binding protein, Sas, using one of these methods, and will characterize Sas's roles in regulation of Ptp10D function in vivo. We have also shown that a signal from glia to neurons is required for axonal expression of a Ptp99A binding protein. We will follow up on these discoveries, and also continue our search for new binding proteins for four different RPTPs. Specific aim 3 describes genetic screens for components of RPTP signaling pathways. We recently defined a remarkable and unique protein trafficking phenotype in embryos lacking both Type III RPTPs, Ptp10D and Ptp4E. Tracheal cells need to make new apical membrane in order to create the lumens of tracheal tubes. In the double mutant embryos, apical proteins accumulate in large intracellular vacuoles (bubbles). Basolateral proteins are localized normally. Our results show that EGFR, Rho family GTPases, and Rab GTPases are involved in generation of this phenotype. We suggest that it occurs because EGFR and Rho activites are upregulated when the RPTPs are absent. This shifts the balance between endocytosis and exocytosis, favoring accumulation of apical proteins in fused endocytic vesicles at the expense of the luminal surface. We will examine whether some of these pathway components are also used for regulation of axon guidance by these RPTPs. We will use the tracheal phenotype as the basis for F1 and F2 genetic screens to find new proteins in the RPTP signaling pathways, and will evaluate the functions of the genes we discover in both neurons and tracheae. Specific aim 4 concerns a new method for identification of in vivo substrates of RPTPs. We will express substrate trap mutants of RPTPs in neurons and tracheae, purify tyrosine-phosphorylated proteins that associate with the traps, and identify these proteins by mass spectrometry. We will then characterize the roles of these putative substrates in the RPTP pathways using genetics, and reconstruct their phosphorylation and dephosphorylation in cell culture.